Peroxisome proliferator activated receptors (PPARs) constitute a subfamily of the nuclear hormone receptors. Three distinct PPARs, termed α, δ (also called β, NUC-1 or FAAR) and γ, each encoded by a separate gene and showing a distinct tissue distribution pattern, have been described [Reviews: Desvergne, B. and Wahli, W., Birkhauser. 1: 142–176 (1994); Green, S., Mutation Res. 333: 101–109 (1995); Schoonjans, K. et al., Biochem. Biophys. Acta. 1302: 93–109 (1996); Schoonjans, K. et al., J. Lipid Res. 37: 907–925 (1996)]. Although it was known that PPARs are activated by a wide variety of chemicals including fibrates, phthalates and fatty acids, PPARs were initially considered orphan receptors, since no direct binding of these compounds to the receptors could be demonstrated.
Activated PPARs heterodimerize with retinoid X receptors (RXRs), another subfamily of nuclear hormone receptors, and alter the transcription of target genes after binding to PPAR response elements (PPREs). A PPRE typically contains a direct repeat of the nuclear receptor hexameric DNA core recognition motif, an arrangement termed DR-1 when recognition motifs are spaced by 1 nucleotide [Schoonjans, K. et al., J. Lipid Res. 37: 907–925 (1996)].
Recently, ligands that induce the transcriptional activity of PPARα (fibrates and leukotriene B4) and γ (prostaglandin J derivatives and thiazolidinediones) have been identified [Devchand, P. R. et al., Nature 384: 39–43 (1996); Kliewer, S. A. et al., Cell 83: 813–819 (1995); Forman, B. M. et al., Cell 83: 803–812 (1995); Lehmann, J. M. et al., J. Biol. Chem. 270: 12953–12956 (1995)].
Numerous PPAR target genes have been identified so far [Review: Schoonjans, K. et al., Biochem. Biophys. Acta. 1302: 93–109 (1996)], and additional target genes continue to be identified [Hertz, R. et al., Biochem. J. 319: 241–248 (1996); Ren, B. et al., J. Biol. Chem. 271: 17167–17173 (1996)]. Because they are activated by various fatty acid metabolites as well as several drugs used in the treatment of metabolic disorders, PPARs are key messengers responsible for the translation of nutritional, pharmacological and metabolic stimuli into changes in gene expression.
PPARγ was the first PPAR for which ligands were identified. In rodents, PPARγ was thought to be confined to adipose tissue. However, low levels of PPARγ expression were detected in other tissues. This led to the suggestion that PPARγ is a key factor triggering adipocyte differentiation, a hypothesis later confirmed [Spiegelman, B. M. and Flier, J. S., Cell 87: 377–389 (1996)]. It is now known that several transcription factors including the nuclear receptor PPARγ (6, 7), the family of CCAATT enhancer binding proteins (C/EBP)1 (8–13) and the basic helix-loop-helix leucine zipper transcription factor ADD1/SREBP1 (14, 15) orchestrate the adipocyte differentiation process (for reviews, see Refs. 1, 3, 16–18).
Two isoforms of PPARγ (PPARγ1 and PPARγ2 that differ by an extra 30 amino acids at the N-terminus) have been identified in mice (Tontonoz, et al. Genes & Development 8:1224–1234 (1994)). Two forms of human PPARγ, γ1 and γ2, have been identified; PPARγ1 has been shown to be the most common form in humans [Mukherjee, R. et al., J. Biol. Chem. 272: 8071–8076 (1997)]. PPARγ2 is expressed at high levels specifically in adipose tissue and is induced early in the course of differentiation of 3T3-L1 preadipocytes to adipocytes. Overexpression and activation of PPARγ protein stimulates adipose conversion in cultured fibroblasts (Tontonoz, et al. Cell 79:1147–1156 (1994)). In addition, PPARγ together with C/EBPα can induce transdifferentiation of myoblasts into adipocytes (19). Activation of PPARγ is sufficient to turn on the entire program of adipocyte differentiation (Lehmann, et al. J. Biol. Chemistry 270:12953–12956 (1995)).
PPREs have been identified in several genes that play crucial roles in adipocyte differentiation, most of them affecting lipid storage and control of metabolism. Examples are fatty acid binding protein (aP2) [Tontonoz, P. et al., Genes Dev. 8: 1224–1234 (1994)], phosphoenolpyruvate carboxykinase (PEPCK) [Tontonoz, P. et al., Mol. Cell. Biol; 15: 351–357 (1995)], Acyl CoA Synthase (ACS) [Schoonjans, K. et al., Eur. J. Biochem. 216: 615–622 (1993); Schoonjans, K. et al., J. Biol. Chem. 270: 19269–19276 (1995)], and lipoprotein lipase (LPL) [Schoonjans, K. et al., EMBO J. 15: 5336–5348 (1996)], all of which are regulated by PPARγ.
Recently, prostaglandin J2 (PGJ2) was shown to be a naturally occurring ligand [Forman, B. M. et al., Cell 83: 803–812 (1995); Kliewer, S. A. et al., Cell 83: 813–819 (1995)] and the anti-diabetic thiazolidinediones (TZDs) [Forman, B. M. et al., Cell 83: 803–812 (1995); Lehmann, J. M. et al., J. Biol. Chem. 270: 12953–12956 (1995)] were shown to be synthetic ligands for PPARγ. The identification of PGJ2 and TZDs as PPARγ ligands corroborates the earlier observation that both prostanoids and TZDs are potent inducers of adipose differentiation programs [Gaillard, D. et al., Biochem. J. 257: 389–397 (1989); Negrel, R. et al., Biochem. J. 257: 399–405 (1989); Forman, B. M. et al., Cell 83: 803–812 (1995); Kliewer, S. A. et al., Cell 83: 813–819 (1995); Aubert, J. et al., FEBS Lett. 397: 117–121 (1996)]. TZDs are currently being developed as insulin sensitizers for the treatment of non-insulin dependent diabetes mellitus (NIDDM) [Reviews: Hulin, B. et al., Current Pharm. Design 2: 85–102 (1996); Saltiel, A. R. and Olefsky, J. M., Diabetes 45: 1661–1669 (1996)]. Interestingly, their relative potency to activate PPARγ in vitro correlates well with their anti-diabetic potency in vivo, suggesting that PPARγ mediates their anti-diabetic effect [Berger, J. et al., Endocrinology 137: 4189–4195 (1996); Willson, T. M. et al., J. Med. Chem. 39: 665–668 (1996)]. These observations define the role of PPARγ in adipose differentiation and a role in glucose and lipid metabolism.
Although many PPARs have been isolated and their cDNAs have been cloned from various species (International Patent publication nos. WO 96/23884, WO96/01430, WO95/11974, Elbrecht, A. et al., Biochem. Biophys. Res. Comm. 224: 431–437 (1996), Greene, M. E. et al., Gene Expression 4: 281–299 (1995), Aperlo, C. et al., Gene 162: 297–302 (1995), Sher, T. et al., Biochemistry 32: 5598–5604 (1993), Isseman, et al. Nature 347:645–650 (1990); Dreyer, et al., Cell 68:879–887 (1992); Gottlicher, et al Proc. Natl. Acad. Sci. USA. 89:4653–4657 (1992); Sher, et al. Biochemistry 32:5598–5604 (1993); and Schmidt, et al. Mol. EndocrinoL 6:1634–16414–8 (1992); Tontonoz, et al. Genes & Development 8:1224–1234 (1994); Kliewer, et al. Proc. Nail. Acad. Sci. 91:7355–7359 (1994); Chen, et al. Biochem. and Biophy. Res. Com. 196:671–677 (1993)), information regarding the regulation of PPAR expression is very limited (see, e.g. Wu, Z. et al., Mol. Cell. Biol. 16(8): 4128–4136 (1996), Zhu, Y. et al., Proc. Natl. Acad. Sci. USA 92: 7921–7925 (1995), Mukherjee, R. et al., J. Steroid Biochem. 51(3/4): 157–166 (1994)). In particular, the regulatory regions controlling the expression of the human PPARγ genes have not yet been disclosed.